摘要
黏质沙雷菌plaS基因编码的磷脂酶A1辅助蛋白S(phospholipase A1 accessory protein S,PlaS)能够显著提高磷脂酶A1在大肠杆菌中的表达活性,但对宿主菌的生长具有抑制作用,且抑制机制尚无相关研究报道。本实验通过对PlaS进行生物信息学分析并对其N端截短系列表达菌株的生长特性进行研究以确定其抑制作用关键区域,并通过扫描电子显微镜观察和流式细胞仪检测来初步探究PlaS对表达宿主菌的抑制机理。结果表明,在PlaS N端分别截短前23、24、25、26、27个氨基酸构建的表达菌株中,dS_(23)P_(28)、dS_(24)P_(28)、dS_(25)P_(28)、dS_(26)P_(28)菌株仍表现出不同程度的生长抑制现象,而dS_(27)P_(28)菌株没有出现生长抑制现象,初步表明PlaS N端的前27个氨基酸为抑制关键区域。扫描电子显微镜和流式细胞仪结果显示,PlaS的表达对大肠杆菌的细胞膜有严重的破坏作用,而N端截短前27个氨基酸的PlaS表达后对大肠杆菌细胞膜的损伤作用消失,这一结果也与生长特性结果一致。本实验结果可为深入研究黏质沙雷菌PlaS功能提供理论参考。
The helper protein encoded by the phospholipase A1 accessory protein S(plaS) gene of Serratia marcescens can significantly improve the expression and activity of phospholipase A1 in Escherichia coli, but it can inhibit the growth of the host strain;however, the underlying mechanism has not been reported. The key gene regions involved in the growth inhibitory effect of PlaS were determined by bioinformatic analysis and the growth characteristics of the expression strains constructed by truncating the sequence encoding 23, 24, 25, 26 and 27 N-terminal amino acids of PlaS(dS23 P28, dS24 P28, dS25 P28, dS26 P28 and dS27 P28), and the inhibitory mechanism was preliminarily investigated by scanning electron microscopy(SEM) and flow cytometry(FCM). The results indicated that dS23 P28, dS24 P28, dS25 P28 and dS26 P28 showed growth inhibition to different extents, while dS27 P28 did not. Preliminary results showed that the first 27 amino acids at the PlaS N-terminal were crucial for its inhibitory effect. The results of SEM and FCM showed that the expressed PlaS caused serious damage to the cell morphology of E. coli, while this effect disappeared after truncation of 27 amino acids from the N-terminal. These results were consistent with the growth characteristics. This study provides a theoretical basis for further studies on the functions of Serratia marcescens PlaS.
作者
甘玉飞
薛正莲
周杰
王芳
王洲
刘艳
GAN Yufei;XUE Zhenglian;ZHOU Jie;WANG Fang;WANG Zhou;LIU Yan(College of Biological and Food Engineering,Anhui Polytechnic University,Wuhu 241000,China;Anhui Engineering Laboratory for Industrial Microbiology Molecular Breeding,Wuhu 241000,China;Anhui Engineering Technology Research Center of Microbial Fermentation,Wuhu 241000,China)
出处
《食品科学》
EI
CAS
CSCD
北大核心
2021年第7期45-51,共7页
Food Science
基金
国家自然科学基金面上项目(31471615)。
关键词
黏质沙雷菌
磷脂酶A1辅助蛋白
大肠杆菌
生长抑制
N端截短
Serratia marcescens
phospholipase A1 accessory protein S
Escherichia coli
growth inhibition
N-terminal truncation
